Actinobacillus actinomycetemcomitans (Aalkpha) is associated with localized juvenile periodontitis and expresses a variety of potential virulence factors. One of these factors, a leukotoxin (ltx), destroys human polymorphonuclear leukocytes and macrophages and may impair the host immune response. ltx expression appears to be influenced by growth phase and environmental conditions but the molecular mechanisms controlling these processes are not known. We are interested in defining the regulatory mechanisms which govern the expression of this putative virulence factor. In the present application, we propose to characterize the promoter region of th ltx gene cluster and to identify the cis and/or transacting factors which influence transcription of the ltx operon. The effects of various environmental stresses on ltx transcription will also be evaluated. In Aim 2, we will compare the regulation of ltx in leukotoxic and non-leukotoxic strains o ascertain if the ltx phenotype is dependent primarily on specific properties of the ltx promoters or upon unique regulatory factors expressed by individual A. actinomycetemcomitans strains. We will also study the function of ORF9.2, a newly identified gene within the ltx operon of strain JP2, and determine if it is involved in the regulation of ltx expression. An understanding of the molecular mechanisms and environmental parameters controlling ltx expression in both highly leukotoxic and non-leukotoxic strains of Aalpha is important and may lead to the development of therapeutic strategies designed to limit ltx expression at infected sites. In addition, the regulatory mechanisms governing ltx expression may also control the production of other factors involved in A. actinomycetemcomitans virulence. Identification of these coordinately regulated factors may elucidate additional factors contributing to A. actinomycetemcomitans pathogenesis.